Cold-sensitive and heat-sensitive mutants of Saccharomyces cerevisiae which become reversibly defective in mitochondrial function at the nonpermissive temperature (18 degrees C or 37 degrees C) have been and will continue to be used in genetic and biochemical analyses to elucidate the sequence of events involved in the growth and differentiation of mitochondrial membranes. The broad goals of the proposed research program are:(1) to determine the extent to which membrane biogenesis depends on an ordered pathway of biosynthetic and assembly steps; (2) to ascertain the cellular location of the genetic determinants controlling these processes; and (3) to define at the molecular level the sequence of biochemical events leading to the formation of fully functional membrane-bound enzyme complexes of the inner mitochondrial membrane. Strains have been constructed which contain at least one cold-sensitive and one heat-sensitive mutation in the same cell. By analyzing these strains for the appearance (or disappearance) of membrane function during shifts from one nonpermissive temperature to the other, it has been possible to demonstrate the existence of an ordered sequence of expression for at least three genes in mitochondrial membrane biogenesis. If such a relationship can be demonstrated for other genes it will be possible to construct an "order of function map" for the assembly of a biological membrane in vivo. Biochemical characterization of the electron transport, energy coupling and general biosynthetic capabilities of the defective mitochondria will be carried out. Specific antisera already prepared against purified mitochondrial enzymes will be used to detect and isolate active and inactive forms of membrane proteins and to identify assembly intermediately.